The principal goal of this project is to extend our understanding of the mechanisms of the sodium, potassium pump (Na,K-ATPase) and the calcium pump (SERCA ATPase). Studies in collaboration with J. Froehlich (U. Md.) are directed at testing the hypothesis that certain of our transient kinetic observations result from oligomeric interactions of these pumps that occur during the catalytic cycle. A dependent hypothesis is that oligomeric interactions contribute to the efficiency of cation transport. Our current evidence suggests that the effective interactions consist of "out-of-phase" free energy transfers between two or more catalytic subunits, and that these interactions are observed when P-type cation pumps are examined in their native state. We have examined the effects of a regulatory protein, phospholamban, on the transient kinetics and oligomeric state of the calcium pumps, SERCA1 and SERCA2a. Activation of cardiac muscle Ca2+-ATPase (SERCA2a) by beta-1-agonists has been shown to increase coupled Ca2+ transport and cardiac sarcoplasmic reticulum (CSR) Ca2+ stores. This results from PKA-dependent phosphorylation of phospholamban (PLB), a small intrinsic membrane protein that will associate similarly with both SERCA1 and SERCA2a, but is normally co-expressed only with SERCA2a. In either case the effect of PLB interaction is to decrease the Vmax and the apparent Ca2+ affinity of these calcium pumps, whereas phosphorylation of phospholamban reverses both of these effects. The mechanism responsible for the increased efficiency of energy utilization resulting from activation of SERCA2a by beta-1 agonists has not been determined. However, saturation transfer EPR measurements of spin-labeled SERCA2a indicate that PLB interaction changes the oligomeric state of SERCA2a to one that is functionally monomeric. Earlier pre-steady state kinetic investigations of CSR Ca2+-ATPase by Froehlich et al also suggests that SERCA1 is an oligomer To account for the faster turnover and higher Vmax in SERCA1 relative to SERCA2a, oligomeric pump interactions were hypothesized to contribute to the rapid phase of E2P hydrolysis in SERCA1 which is absent in SERCA2a. We have now tested whether removal of SERCA2a regulation by PLB converts it to a functionally oligomeric state resembling SERCA1. SERCA2a was expressed with (+) or without (-) PLB in High Five insect cells using recombinant baculovirus (provided by the Mahaney lab) Quenched-flow mixing was used to measure the kinetics of EP formation at different ATP concentrations; dephosphorylation was measured by chasing the steady state EP with ADP or EGTA. Rate parameters for the quenched-flow data were evaluated by means of an iterative curve-fitting routine that fit the data to a sum of exponentials. In rapid mixing experiments, SERCA2a + PLB behaved like native cardiac SR Ca2+-ATPase in demonstrating kinetic properties resembling those of a monomer. These include the absence of stimulation by ATP (10-50 mM) of pre-steady EP overshoot decay, a small (9%) steady state fraction of E1P and the absence of a rapid phase of EGTA-induced EP decay. In contrast, SERCA2a sans PLB showed two-fold stimulation of EP overshoot decay by ATP, a larger (31%) steady state fraction of E1P and the presence of a rapid initial phase of EGTA-induced EP decay. These characteristics are similar to those seen in skeletal SR Ca2+-ATPase and suggest that SERCA2a sans PLB, like SERCA1, functions as an oligomer. In parallel experiments the Mahaney lab has found that spin-labeled SERCA2a co-expressed with PLB has a smaller rotational correlation time (63 ms) than either SERCA2a sans PLB (78 ms) or SERCA2a + phosphorylated-PLB (97 ms) prepared by incubation with MgATP in the presence of cAMP-dependent PKA and phosphatase inhibitor. Thus the rotational correlation times also suggest a more compact structure for SERCA2a + PLB (monomer or compact dimer?) than for SERCA2a sans PLLB or SERCA2a + phosphorylated-PLB (less compact dimer?). We conclude that either genetic deletion of PLB or its phosphorylation by PKA can convert SERCA2a from a monomer (or uncoupled oligomer) to a coupled oligomer, which exhibits an improved Ca2+ pumping efficiency through inter-subuint free energy exchange. Some of this work was presented at the 2002 International Conference on Na,K-ATPases and Related Cation Pumps. The manuscript covering this work has been submitted for publication.